The hematological process leading to the production and maturation of red blood cells is under the control of erythropoietin (EPO) (reviewed in Krantz, S. B. (1991) Erythropoietin. Blood 77, 419-434), a glycoprotein hormone primarily synthesized in the kidney. Commercially available human EPO is produced via recombinant DNA techniques and is known as recombinant human EPO (rhEPO). rhEPO has a molecular mass of approximately 36,000 Daltons, as determined by SDS-PAGE. The molecular mass of the protein backbone is 18,398 Daltons, which indicates that the entire molecule is heavily glycosylated. The carbohydrate residues are important for in in vivo biologic activity.
In contrast to many other growth factors, the specificity of EPO for erythroid cells has lead to its development as a safe and efficacious therapeutic protein. The medical benefits of EPO have been well established in the treatment of anemia associated with chronic renal failure, cancer chemotherapy, and autologous predonation of blood. Due to the chronic nature of EPO therapy, it would be desirable to have an orally administered "second generation" molecule.
An understanding of the structural basis of the interaction of EPO with its receptor will aid in the design of new drugs, such as an oral anemia drug. Traditional methods of drug discovery are being supplemented by rational design approaches that attempt to make use of information about the structural basis of receptor-ligand interactions to develop molecular models. These models, in turn, are used to design molecules with therapeutic potential. The two approaches are complementary and rely on the ability to obtain information about the three-dimensional structure of the therapeutic target. The ability to produce EPO and its receptor and to assess the impact of structural changes on protein function provides a means for testing hypothetical molecular models and contributes to the establishment of a structure activity relationship database for drug design.
The biological effect of EPO appears to be mediated, in part, through interaction with a cell membrane bound receptor which has previously been cloned (Jones, S. S., D'Andrea, A. D., Haines, L. L., and Wong, G. G. (1990), Human erythropoietin receptor: Cloning, expression, and biological characterization, Blood 76, 31-35; Noguchi, C. T., Kyung, S. B., Chin, K., Wada, Y., Schecter, A. N. and Hankins, W. D. (1991) Cloning of the human erythropoietin receptor gene, Blood 78, 2548-2556; Maouche, L., Tournamile C., Hattab, C., Boffa, G., Carton J.-P. and Chretein, S. (1991) Cloning of the gene encoding the human erythropoietin receptor. Blood 78, 2557-2563). Currently there is considerable interest in the physical nature of the association of EPO with the EPO receptor (EPOR) and an emerging technique for the analysis of this type of interaction is the generation of soluble receptors, also termed hormone binding proteins (Langer, J. A. (1990) Soluble ligand-binding fragments of polypeptide receptors in basic and applied research, Pharmaceutical Technology 14, 46-66). The process involves the engineering of suitable expression vectors encoding the extracellular domain of the receptor and the subsequent production and purification of the protein. Once obtained in active form, these soluble receptor fragments are useful in numerous assay formats and have improved utility in biophysical studies such as NMR or X-ray crystallography, since they can be employed in conditions free of the detergents required to solubilize membrane bound receptors. Some receptor fragments of this type, including IL-1 and IL-4, function to neutralize the biological effects of the hormone and appear to have therapeutic potential (Maliszewski, C. R. and Fanslow, W. C. (1990) Soluble receptors for IL-1 and IL-4: Biological activity and therapeutic potential, Trends in Biotech 8, 324-329).
Previous reports outline the development of systems for the production and purification of human and murine EPO binding protein (EBP) utilizing protein expression in eukaryotic cells and bacteria but with modest yields (Harris, K. W., Mitchell, R. A., and Winkleman, J. C. (1992) Ligand Binding Properties of the Human Erythropoietin Receptor Extracellular Domain Expressed in E. coli. J. Biol. Chem. 267, 15205-15209; Yet, M.-G and Jones, S. S. (1993) The Extracytoplasmic Domain of the Erythropoietin Receptor Forms a Monomeric Complex with Erythropoietin. Blood 82, 1713-1719; Nagao, M., Masuda, S., Abe, S., Ueda, M. and Sasaki, R. (1992) Production and ligand-binding characteristics of a soluble form of the murine erythropoietin receptor, Biochem. Biophys. Res. Comm. 188, 888-897).
The mechanism of EPO receptor activation has been suggested to reside in the dimerization of two EPO receptor molecules which results in subsequent steps of signal transduction [Watowich, S. S., Yohimura, A., Longmore, G. D., Hilton, D. J., Yoshimura, and Lodish, H. F., Homodimerization and constitutive activation of the erythropoietin receptor. Proceedings of the National Academy of Sciences 89, 2140-2144 (1992)]. While the soluble EPO receptor [Johnson, D. L., Middleton, S. A., McMahon, F., Barbone, F., Kroon, D., Tsao, E., Lee, W. H., Mulcahy, L. S. and Jolliffe, L. K., Refolding, Purification and Characterization of Human Erythropoietin Binding Protein Produced in Escherichia coli, Protein Expression and Purification 7 104-113 (1996)] has advantages related to structure determination and ease of production, it likely does not represent a preformed template for receptor dimerization. In the search for peptides or small molecules which might bind to and activate the EPO receptor such a preformed dimerization template is a highly valuable tool for the discovery, detection, and description of molecules with such activity. Use of receptor-Ig fusion molecules provide such templates and depending upon the assay format provide information on the ability of a given molecule or compound to detect non-productive as well as productive dimerization complexes.